Oligonucleotides having chiral phosphorus linkages

ABSTRACT

Sequence-specific oligonucleotides are provided having substantially pure chiral Sp phosphorothioate, chiral Rp phosphorothioate, chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate, chiral Rp phosphoamidate, chiral Sp phosphotriester, and chiral Rp phosphotriester linkages. The novel oligonucleotides are prepared via a stereospecific SN 2  nucleophilic attack of a phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate anion on the 3′ position of a xylonucleotide. The reaction proceeds via inversion at the 3′ position of the xylo reactant species, resulting in the incorporation of phosphodiester, phosphorothioate, phosphoramidate, phosphotriester or alkylphosphonate linked ribofuranosyl sugar moieties into the oligonucleotide.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 91/00243, filed Jan. 11, 1991, which is a continuation-in-partof application Ser. No. 463,358 filed Jan. 11, 1990 and of applicationSer. No. 566,977, filed Aug. 13, 1990. The entire disclosures of bothapplications, which are assigned to the assignee of this invention, areincorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention is directed to sequence-specific oligonucleotideshaving chiral phosphorus linkages and to a novel chemical synthesis ofthese and other oligonucleotides. The invention includes chiralalkylphosphonate, chiral phosphotriester, and chiralphosphoramidate-linked oligonucleotides. The invention further includeschiral phosphorothioate, chiral alkylphosphonate, chiralphosphotriester, and chiral phosphoramidate-linked oligonucleotides thatcontain at least one modified nucleoside unit. The novel chemicalsynthesis provides such chiral phosphorothioate, chiralalkylphosphonate, chiral phosphotriester, and chiral phosphoramidateoligonucleotides as well as “natural” or “wild type” phosphodiesteroligonucleotides.

BACKGROUND OF THE INVENTION

[0003] Messenger RNA (mRNA) directs protein synthesis. As a therapeuticstrategy, antisense therapy strives to disrupt the synthesis of targetproteins by using a sequence-specific oligonucleotide to form a stableheteroduplex with its corresponding mRNA. Such antisenseoligonucleotides generally have been natural phosphodiesteroligonucleotides.

[0004] As contrasted to natural phosphodiester oligonucleotides, the useof phosphorothioate, methylphosphonate, phosphotriester orphosphoramidate oligonucleotides in antisense therapy provides certaindistinguishing features. Each of the phosphorothioate,methylphosphonate, phosphotriester or phosphoramidate phosphoruslinkages can exist as diastereomers. Certain of these phosphorothioate,methylphosphonate, phosphotriester or phosphoramidate oligonucleotideshave a greater resistance to nucleases. Some have solubilities similarto the solubility of natural phosphodiester oligonucleotides. Other havesolubilities different from that of the natural phosphodiesteroligonucleotides. Some are generally more chemically orthermodynamically stable than the natural phosphodiesteroligonucleotides. At least the phosphorothioates haveoligonucleotide-RNA heteroduplexes that can serve as substrates forendogenous RNase H.

[0005] The phosphorothioate oligonucleotides, like the naturalphosphodiester oligonucleotides, are soluble in aqueous media. Incontrast, methylphosphonate, phosphotriester, and phosphoramidateoligonucleotides, which lack a charge on the phosphorus group, canpenetrate cell membranes to a greater extent and, thus, facilitatecellular uptake. The internucleotide linkage in methylphosphonateoligonucleotides is more base-labile than that of the naturalphosphodiester internucleotide linkage, while the internucleotidelinkage of the phosphorothioate oligonucleotides is more stable than thenatural phosphodiester oligonucleotide linkage.

[0006] The resistance of phosphorothioate oligonucleotides to nucleaseshas been demonstrated by their long half-life in the presence of variousnucleases relative to natural phosphodiester oligonucleotides. Thisresistance to nucleolytic degradation in vitro also applies to in vivodegradation by endogenous nucleases. This in vivo stability has beenattributed to the inability of 3′-5′ plasma exo-nucleases to degradesuch oligonucleotides. Phosphotriester and methylphosphonateoligonucleotides also are resistant to nuclease degradation, whilephosphoramidate oligonucleotides show some sequence dependency.

[0007] Since they exist as diastereomers, phosphorothioate,methylphosphonate, phosphotriester or phosphoramidate oligonucleotidessynthesized using known, automated techniques result in racemic mixturesof Rp and Sp diastereomers at the individual phosphorothioate,methylphosphonate, phosphotriester or phosphoramidate linkages. Thus, a15-mer oligonucleotide containing 14 asymmetric linkages has 2⁴¹, i.e.16,384, possible stereoisomers. Accordingly, it is possible that only asmall percentage of the oligonucleotides in a racemic mixture willhybridize to a target mRNA or DNA with sufficient affinity to proveuseful in antisense or probe technology.

[0008] Miller, P. S., McParland, K. B., Jayaraman, K., and Ts'o, P. O. P(1981), Biochemistry, 20:1874, found that small. di-, tri- andtetramethylphosphonate and phosphotriester oligonucleotides hybridize tounmodified strands with greater affinity than natural phosphodiesteroligonucleotides. Similar increased hybridization was noted for smallphosphotriester and phosphoramidate oligonucleotides; Koole, L. H., vanGenderen, M. H. P., Reiners, R. G., and Buck, H. M. (1987), Proc. K.Ned. Adad. Wet., 90:41; Letsinger, R. L., Bach, S. A., and Eadie, J. S.(1986), Nucleic Acids Res., 14:3487; and Jager, A., Levy, M. J., andHecht, S. M. (1988), Biochemistry, 27:7237. The effects of the racemicdiastereomers on hybridization becomes even more complex as chain lengthincreases.

[0009] Bryant, F. R. and Benkovic, S. J. (1979), Biochemistry, 18:2825studied the effects of diesterase on the diastereomers of ATP. Publishedpatent application PCT/US88/03634 discloses dimers and trimers of2′-5′-linked diastereomeric adenosine units. Niewiarowski, W.,Lesnikowski, Z. J., Wilk, A., Guga, P., Okruszek, A., Uznanski, B., andStec, W. (1987), Acta Biochimica Polonia, 34:217, synthesizeddiastereomeric dimers of thymidine, as did Fujii, M., Ozaki, K., Sekine,M., and Hata, T. (1987), Tetrahedron, 43:3395.

[0010] Stec, W. J., Zon, G., and Uznanski, B. (1985), J. Chromatography,326:263, have reported the synthesis of certain racemic mixtures ofphosphorothioate or methyphosphonate oligonucleotides. However, theywere only able to resolve the diastereomers of certain small oligomershaving one or two diastereomeric phosphorus linkages.

[0011] In a preliminary report, J. W. Stec, Oligonucleotides asantisense inhibitors of gene expression: Therapeutic implications,meeting abstracts, Jun. 18-21, 1989, noted that a non-sequence-specificthymidine homopolymer octomer—i.e. a (dT)₈-mer, having “all-except-one”Rp configuration methylphosphonate linkages—formed a thermodynamicallymore stable hybrid with a 15-mer deoxyadenosine homopolymer—i.e. ad(A)₁₅-mer—than did a similar thymidine homopolymer having“all-except-one” Sp configuration methylphosphonate linkages. The hybridbetween the “all-except-one” Rp (dT)₈-mer and the d(A)₁₅-mer had a Tm of38° .C while the Tm of the “all-except-one” Sp (dT)₈-mer and thed(A)₁₅-mer was <0° C. The hybrid between a (dT)₈-mer having naturalphosphodiester linkages, i.e. octathymidylic acid, and the d(A)₁₅-merwas reported to have a Tm of 14° C. The “all-except-one” thymidinehomopolymer octamers were formed from two thymidine methylphosphonatetetrameric diastereomers linked by a natural phosphodiester linkage.

[0012] To date, it has not been possible to chemically synthesize anoligonucleotide having more than two adjacent, chirally pure phosphorouslinkages. Indeed, even in homopolymers it has been possible to produceonly three such adjacent chiral linkages. For an oligonucleotide to beuseful as an antisense compound, many nucleotides must be present. Whilenot wishing to be bound by any particular theory, it is presentlybelieved that generally at least about 10 or more nucleotides arenecessary for an oligonucleotide to be of optimal use as an antisensecompound. Because it has not been possible to resolve more than two orthree adjacent phosphorus linkages, the effects of induced chirality inthe phosphorus linkages of chemically synthesized antisenseoligonucleotides has not been well assessed heretofore.

[0013] Except as noted above, the sequence-specific phosphorothioate,methylphosphonate, phosphotriester or phosphoramidate oligonucleotidesobtained utilizing known automated synthetic techniques have beenracemic mixtures. Indeed, it was recently stated in a review articlethat: “It is not yet possible to synthesize by chemical meansdiastereomerically pure chains of the length necessary for antisenseinhibition,” see J. Goodchild (1990) Bioconjugate Chemistry, 1:165.

[0014] The use of enzymatic methods to synthesize oligonucleotideshaving chiral phosphorous linkages has also been investigated. Burgers,P. M. J. and Eckstein, F. (1979), J. Biological Chemistry, 254:6889; andGupta, A., DeBrosse, C., and Benkovic, S. J. (1982) J. Bio. Chem.,256:7689 enzymatically synthesized diastereomeric polydeoxyadenylic acidhaving phosphorothioate linkages. Brody, R. S. and Frey, P. S. (1981),Biochemistry, 20:1245; Eckstein, F. and Jovin, T. M. (1983),Biochemistry, 2:4546; Brody, R. S., Adler, S., Modrich, P., Stec, W. J.,Leznikowski, Z. J., and Frey, P. A. (1982) Biochemistry, 21: 2570-2572;and Romaniuk, P. J. and Eckstein, F. (1982) J. Biol. Chem.,257:7684-7688 all enzymatically synthesized poly TpA and poly ApTphosphorothioates while Burgers, P. M. J. and Eckstein, F. (1978) Proc.Natl. Acad. Sci. USA, 75: 4798-4800 enzymatically synthesized poly UpAphosphorothioates. Cruse, W. B. T., Salisbury; T., Brown, T., Cosstick,R. Eckstein, F., and Kennard, O. (1986), J. Mol. Biol., 192:891, linkedthree diastereomeric Rp GpC phosphorothioate dimers via naturalphosphodiester bonds into a hexamer. Most recently Ueda, T., Tohda, H.,Chikazuni, N., Eckstein, R., and Watanabe, K. (1991) Nucleic AcidsResearch, 19:547, enzymatically synthesized RNA's having from severalhundred to ten thousand nucleotides incorporating Rp diastereomericphosphorothioate linkages. Enzymatic synthesis, however, depends on theavailability of suitable polymerases that may or may not be available,especially for modified nucleoside precursors.

[0015] Thus, while phosphorothioate, alkylphosphonate, phosphoamidate,and phosphotriester oligonucleotides have useful characteristics, littleis known concerning the effects of differing chirality at the phosphoruslinkages. It would therefore be of great advantage to provideoligonucleotides having phosphorous linkages of controlledstereochemistry.

OBJECTS OF THE INVENTION

[0016] Accordingly, it is one object of this invention to providesequence-specific oligonucleotides having chirally purephosphorothioate, alkylphosphonate, phosphotriester or phosphoramidatelinkages.

[0017] It is a further object to provide phosphorothioate,alkylphosphonate, phosphoramidate, and phosphotriester oligonucleotidescomprising substantially all Rp or all Sp linkages.

[0018] It is another object to provide phosphorothioate,alkylphosphonate, phosphoramidate, and phosphotriester oligonucleotidesthat have antisense hybridizability against DNA and RNA sequences.

[0019] It is still another object of this invention to providephosphorothioate, alkylphosphonate, phosphoramidate, and phosphotriesteroligonucleotides for use in antisense diagnostics and therapeutics.

[0020] A further object is to provide research and diagnostic methodsand materials for assaying bodily states in animals, especially diseasedstates.

[0021] Another object is to provide therapeutic and research methods andmaterials for the treatment of diseases through modulation of theactivity of DNA and RNA.

[0022] It is yet another object to provide new methods for synthesizingsequence-specific oligonucleotides having chirally purephosphorothioate, methylphosphonate, phosphotriester or phosphoramidatelinkages.

SUMMARY OF THE INVENTION

[0023] The present invention provides stereoselective methods forpreparing sequence-specific oligonucleotides having chiral phosphorouslinkages. In certain preferred embodiments, these methods comprise thesteps of:

[0024] (a) selecting a first synthon having structure (1):

[0025] wherein:

[0026] Q is O or CH₂;

[0027] R_(A) and R_(B) are H, lower alkyl, substituted lower alkyl, anRNA cleaving moiety, a group which improves the pharmacokineticproperties of an oligonucleotide, or a group which improves thepharmacodynamic properties of an oligonucleotide;

[0028] R_(D) is O, S, methyl, alkoxy, thioalkoxy, amino or substitutedamino;

[0029] R_(E) is O or S;

[0030] R_(X) is H, OH, or a sugar derivatizing group;

[0031] B_(X) is a naturally occurring or synthetic nucleoside base orblocked nucleoside base; and

[0032] Y is a stable blocking group, a solid state support, a nucleotideon a solid state support, or an oligonucleotide on a solid statesupport;

[0033] (b) selecting a second synthon having structure (2):

[0034] wherein:

[0035] R_(F) is a labile blocking group; and

[0036] L is a leaving group or together L and Bx are a 2-3′ or 6-3′pyrimidine or 8-3′ purine cyclonucleoside;

[0037] (c) adding the second synthon to the first synthon in thepresence of a base to effect nucleophilic attack of the 5′-phosphate ofthe first synthon at the 3′-position of the second synthon to yield anew first synthon having structure (3):

[0038] via a stereospecific inversion of configuration at the 3′position of the second synthon; and

[0039] (d) treating the new first synthon with a reagent to remove thelabile blocking group R_(F).

[0040] Additional nucleotides are added to the new first synthon byrepeating steps (b), (c), and (d) for each additional nucleotide.Preferably, R_(F) is an acid-labile blocking group and said new firstsynthon in step (d) is treated with an acidic reagent to remove saidacid-labile R_(F) blocking group.

[0041] The present invention also provides sequence-specificoligonucleotides comprising a plurality of nucleotides linked by chiralphosphorothioate, methylphosphonate, phosphotriester or phosphoramidateoligonucleotides linkages wherein at least one of the nucleosides is anon-naturally occurring nucleoside. Preferably, the nucleosides areconnected via linkages selected from the group consisting of chiral Spphosphorothioate,, chiral Rp phosphorothioate, chiral Spalkylphosphonate, chiral Rp alkylphosphonate, chiral Sp phosphoamidate,chiral Rp phosphoamidate, chiral Sp chiral phosphotriester or chiral Rpphosphotriester linkages. In one embodiment of the invention each of thelinkages of the oligonucleotide is a substantially pure chiralphosphorous linkage. In other embodiments less than all of the phosphatelinkages are substantially pure chiral phosphorous linkages. In furtherembodiments, the oligonucleotides of the invention form at least aportion of a targeted RNA or DNA sequence.

[0042] The present invention also provides oligonucleotides comprisingnucleoside units joined together by either all Sp phosphotriesterlinkages, all Rp phosphotriester linkages, all Sp phosphoramidatelinkages, or all Rp phosphoramidate linkages. Also provided areoligonucleotides having at least 10 nucleoside units joined together byeither all Sp alkylphosphonate linkages or all Rp alkylphosphonatelinkages. Preferably such alkylphosphonate linkages aremethylphosphonate linkages. Each of these oligonucleotides can form atleast a portion of a targeted RNA or DNA sequence.

[0043] In preferred embodiments of the invention, the oligonucleotidesinclude non-naturally occurring nucleoside units incorporated into theoligonucleotide chain. Such nucleoside units preferably have structure(4) or structure (5):

[0044] wherein Q, R_(B), R_(G), and B_(X) are defined as above and R_(C)is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF₃,OCF₃, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substituted S-alkyl,SOMe, SO₂Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl,OCH₂CH═CH₂, OCH═CH₂, OCH₂CCH, OCCH, aralkyl, heteroaralkyl,heterocycloalkyl, poly-alkylamino, substituted silyl, an RNA cleavingmoiety, a group which improves the pharmacodynamic properties of anoligonucleotide, or a group which improves the pharmacokineticproperties of an oligonucleotide.

[0045] In preferred embodiments, B_(X) is a pyrimidinyl-1 or purinyl-9moiety such as, for example, adenine, guanine, hypoxanthine, uracil,thymine, cytosine, 2-aminoadenine or 5-methylcytosine.

[0046] In further preferred embodiments, the modified nucleosidesinclude nucleosides having structures (6)-(11):

[0047] wherein:

[0048] G and K are, independently, C or N;

[0049] J is N or CR₂R₃;

[0050] R₁ is OH or NH₂;

[0051] R₂ and R₃ are H, NH, lower alkyl, substituted lower alkyl, loweralkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino,heterocycloalkyl, heterocycloalkylamino, aminoalkylamino,hetrocycloalkylamino, polyalkylamino, an RNA cleaving moiety, a groupwhich improves the pharmacokinetic properties of an oligonucleotide, ora group which improves the pharmacodynamic properties of anoligonucleotide;

[0052] R₄ and R₅ are, independently, H, OH, NH₂, lower alkyl,substituted lower alkyl, substituted amino, an RNA cleaving moiety, agroup which improves the pharmacokinetic properties of anoligonucleotide, or a group which improves the pharmacodynamicproperties of an oligonucleotide;

[0053] R₆ and R₇ are, independently, H, OH, NH₂, SH, halogen, CONH₂,C(NH)NH₂, C(O)O-alkyl, CSNH₂, CN, C(NH)NHOH, lower alkyl, substitutedlower alkyl, substituted amino, an RNA cleaving moiety, a group whichimproves the pharmacokinetic properties of an oligonucleotide, or agroup which improves the pharmacodynamic properties of anoligonucleotide; and

[0054] X is a sugar or a sugar analog moiety where said sugar analogmoiety is a sugar substituted with at least one substituent comprisingan RNA cleaving moiety, a group which improves the pharmacodynamicproperties of an oligonucleotide, or a group which improves thepharmacokinetic properties of an oligonucleotide.

[0055] The present invention also provides compounds which are useful informing the oligonucleotides of invention. Such compounds have structure(12):

[0056] wherein Q, R_(A), R_(D),R_(E), R_(X), L, and B_(X) are defined asabove and R_(F) is H or a labile blocking group.

[0057] The oligonucleotides of the invention are useful to increase thethermodynamic stability of heteroduplexes with target RNA and DNA.Certain of the oligonucleotides of the invention are useful to elicitRNase H activity as a termination event. Certain other oligonucleotidesare useful to increase nuclease resistance. The oligonucleotides of theinvention are also useful to test for antisense activity using reportergenes in suitable assays and to test antisense activity against selectedcellular target mRNA's in cultured cells.

DETAILED DESCRIPTION OF THE INVENTION

[0058] As will be recognized, adjacent nucleosides of a naturallyoccurring or wild type oligonucleotide are joined together byphosphodiester linkages, i.e. diesters of phosphoric acid. The naturalphosphodiester linkages in oligonucleotides are at the same time bothnon-chiral and pro-chiral sites. Substitution of one of the oxygen atomsof the phosphate moiety of a nucleotide with another atom yields anasymmetric center on the phosphorus atom. Since a nucleotide unitalready contains a first asymmetrical center within its sugar moiety,further asymmetry at the phosphorus atom of the nucleotide yields adiasymmetric nucleotide. Such a diasymetric nucleotide is a chiralnucleotide having Sp and Rp diastereomers.

[0059] Substitution of one of the oxygen atoms of the phosphate moietyof a nucleotide with a sulfur atom yields Sp and Rp diastereomericphosphorothioate analogs. Similarly, substitution of a phosphate oxygenatom by an alkyl moiety yields diastereomeric alkylphosphonate analogs.Substitution with an alkoxy group yields diastereomeric Sp and Rpphosphotriesters. Substitution with a thioalkoxy group yields a mixedtriester—a phosphodiesterthioester. Substitution with an amine or asubstituted amine (including heterocyclic amines) yields diastereomericSp and Rp phosphoramidates.

[0060] It will be appreciated that the terms “phosphate” and “phosphateanion” as employed in connection with the present invention includenucleotides and oligonucleotides derived by replacement of one of theoxygen atoms of a naturally occurring phosphate moiety with aheteroatom, an alkyl group or an alkyoxy group. Thus, the terms“phosphate” or “phosphate anion” include naturally occurringnucleotides, phosphodiesters of naturally occurring oligonucleotides, aswell as phosphorothioate, alkylphosphonate, phosphotriester, andphosphoamidate oligonucleotides.

[0061] Since there exist numerous phosphodiester linkages in anoligonucleotide, substitution of an oxygen atom by another atom such as,for example, sulfur, nitrogen, or carbon in one or more of the phosphatemoieties yields a racemic mixture unless such substitution occurs in astereospecific manner. As a practical matter, see Stec, W. J., Zon, G.,and Uznanski, B. (1985), J. Chromatography, 326:263, above. Separationof the diastereomers of racemic mixtures of non-stereospecificsynthesized oligonucleotides is only possible when there are a minimumof diasymmetric sites, for example, two diasymmetric sites. Since thediasymmetric substituent group at each diastereomeric phosphorus atomcould have steric, ionic or other effects on conformation, binding, andthe like at each such site, sequence-specific oligonucleotides havingall Sp or all Rp chiral phosphorus linkages are desirable.

[0062] In accordance with this invention, sequence-specificoligonucleotides are provided comprising substantially pure chiralphosphate linkages such as, for example, phosphorothioate,methylphosphonate, phosphotriester or phosphoramidate linkages. Incontrast to prior art synthetic oligonucleotides, at least certain ofthe chiral phosphorous linkages of the present oligonucleotides are notracemic in nature but, rather, possess relatively high enantiomericpurity. As will be recognized by those skilled in the art, enantiomericpurity —also known as chiral purity—is manifested for a chemicalcompound by the predominance of one enantiomer over the other. Thus, anoligonucleotide can be said to possess a substantially pure chiralphosphate linkage where, for example, the Sp form of that linkagegreatly predominates over the Rp form. In accordance with the presentinvention, at least certain of the chiral phosphate linkages present inan oligonucleotide should have chiral purity greater than about 75%.Preferably such linkages have chiral purity greater than about 90%, morepreferably greater than about 95%, even more preferably about 100%.Chiral purity may be determined by any of the many methods known in theart, including but not limited to x-ray diffraction, optical rotarydispersion, and circular dichroism.

[0063] The oligonucleotides of the invention are expected to exhibit oneor more efficacious properties such as, for example, hybridization withtargeted RNA's and DNA's, cellular absorption and transport, or improvedenzymatic interaction. At the same time, it is expected that theseimprovements to the basic oligonucleotide sequences will notsignificantly diminish existing properties of the basic oligonucleotidesequence. Thus, the present improvements are likely to lead to improveddrugs, diagnostics, and research reagents.

[0064] Further improvements likely can be effected by making one or moresubstitutions or modifications to the base or the sugar moieties of theindividual nucleosides employed to prepare the chiral oligonucleotidesof the invention. Such substitutions or modifications generally comprisederivation at a site on the nucleoside base or at a site on thenucleoside sugar, provided such derivation does not interfere with thestereoselective syntheses of the present invention by, for example,blocking nucleophilic attack of the 5′-phosphate of a first synthon atthe 3′-position of a second synthon. In certain embodiments, one or moreof the nucleosides of the chiral oligonucleotides of the inventioninclude a naturally occurring nucleoside unit which has been substitutedor modified. These non-naturally occurring or “modified” nucleosideunits preferably have either structure (4) or structure (5):

[0065] wherein:

[0066] Q is O or CHR_(G);

[0067] R_(A) and R_(B) are H, lower alkyl, substituted lower alkyl, anRNA cleaving moiety, a group which improves the pharmacokineticproperties of an oligonucleotide, or a group which improves thepharmacodynamic properties of an oligonucleotide;

[0068] R_(C) is H, OH, lower alkyl, substituted lower alkyl, F, Cl, Br,CN, CF₃, OCF₃, OCN, O-alkyl, substituted O-alkyl, S-alkyl, substitutedS-alkyl, SOMe, SO₂Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substitutedNH-alkyl, OCH₂CH═CH₂, OCH═CH₂, OCH₂CCH, OCCH, aralkyl, heteroaralkyl,heterocycloalkyl, polyalkylamino, substituted silyl, an RNA cleavingmoiety, a group which improves the pharmacodynamic properties of anoligonucleotide, or a group which improves the pharmacokineticproperties of an oligonucleotide;

[0069] R_(G) is H, lower alkyl, substituted lower alkyl, an RNA cleavingmoiety, a group which improves the pharmacokinetic properties of anoligonucleotide, or a group which improves the pharmacodynamicproperties of an oligonucleotide; and

[0070] B_(X) is a nucleoside base, a blocked nucleoside base, anucleoside base analog, or a blocked nucleoside base analog.

[0071] In preferred embodiments B_(X) is a pyrimidinyl-1 or purinyl-9moiety as for instance adenine, guanine, hypoxanthine, uracil, thymine,cytosine, 2-aminoadenine or 5-methylcytosine. Preferably, B_(X) isselected such that a modified nucleoside has one of the structures(6)-(11):

[0072] wherein:

[0073] G and K are, independently, C or N;

[0074] J is N or CR₂R₃;

[0075] R₁ is OH or NH₂;

[0076] R₂ and R₃ are H, NH, lower alkyl, substituted lower alkyl, loweralkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino,heterocycloalkyl, heterocycloalkylamino, aminoalkylamino,hetrocycloalkylamino, polyalkylamino, an RNA cleaving moiety, a groupwhich improves the pharmacokinetic properties of an oligonucleotide, ora group which improves the pharmacodynamic properties of anoligonucleotide;

[0077] R₄ and R₅ are, independently, H, OH, NH₂, lower alkyl,substituted lower alkyl, substituted amino, an RNA cleaving moiety, agroup which improves the pharmacokinetic properties of anoligonucleotide, or a group which improves the pharmacodynamicproperties of an oligonucleotide;

[0078] R₆ and R₇ are, independently, H, OH, NH₂, SH, halogen, CONH₂,C(NH)NH₂, C(O)O-alkyl, CSNH₂, CN, C(NH)NHOH, lower alkyl, substitutedlower alkyl, substituted amino, an RNA cleaving moiety, a group whichimproves the pharmacokinetic properties of an oligonucleotide, or agroup which improves the pharmacodynamic properties of anoligonucleotide; and

[0079] X is a sugar or a sugar analog moiety where said sugar analogmoiety is a sugar substituted with at least one substituent comprisingan RNA cleaving moiety, a group which improves the pharmacodynamicproperties of an oligonucleotide, or a group which improves thepharmacokinetic properties of an oligonucleotide. It is preferred that Xhave the general structure (4) or (5).

[0080] For the purposes of this invention, improving pharmacodynamicproperties means improving oligonucleotide uptake, enhancedoligonucleotide resistance to degradation, and/or strengthenedsequence-specific hybridization with RNA and improving pharmacokineticproperties means improved oligonucleotide uptake, distribution,metabolism or excretion. RNA cleaving moieties are chemical compounds orresidues which are able to cleave an RNA strand in either a random or,preferably, a sequence-specific fashion.

[0081] Exemplary base moieties of the invention are any of the naturalpyrimidinyl-1- or purinyl-9- bases including uracil, thymine, cytosine,adenine, guanine, 5-alkylcytosines such as 5-methylcytosine,hypoxanthine, 2-aminoadenine, and other modified bases as depicted inthe formulas above. Exemplary sugars include ribofuranosyl,2′-deoxyribofuranosyl, their corresponding five membered ringcarbocyclic analogs as well as other modified sugars depicted in theformulas above. Particularly preferred modified sugars include 2′-fluoroand 2′-O-methyl-2′-deoxyribofuranosyl, i.e. 2′-fluoro and2′-O-methyl-β-D-erythro-pentofuranosyl.

[0082] Alkyl groups of the invention include but are not limited toC₁-C₁₂ straight and branched chained alkyls such as methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, isopropyl, 2-butyl, isobutyl, 2-methylbutyl, isopentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl. Alkenylgroups include but are not limited to unsaturated moieties derived fromthe above alkyl groups including but not limited to vinyl, allyl,crotyl, propargyl. Aryl groups include but are not limited to phenyl,tolyl, benzyl, naphthyl, anthracyl, phenanthryl, and xylyl. Halogensinclude fluorine, chlorine, bromine, and iodine. Suitable heterocyclicgroups include but are not limited to imidazole, tetrazole, triazole,pyrrolidine, piperidine, piperazine, and morpholine. Carbocyclic groupsinclude 3, 4, 5, and 6-membered substituted and unsubstituted alkyl andalkenyl carbocyclic rings. Amines include amines of all of the abovealkyl, alkenyl, and aryl groups including primary and secondary aminesand “masked amines” such as phthalimide. Amines are also meant toinclude polyalkylamino compounds and aminoalkylamines such asaminopropylamine and further heterocyclo-alkylamines such as imidazol-1,2 or 4-yl-propylamine. Substituent groups for the above include but arenot limited to other alkyl, haloalkyl, alkenyl, alkoxy, thioalkoxy,haloalkoxy, and aryl groups as well as halogen, hydroxyl, amino, azido,carboxy, cyano, nitro, mercapto, sulfides, sulfones, and sulfoxides.Other suitable substituent groups also include rhodamines, coumarins,acridones, pyrenes, stilbenes, oxazolo-pryidocarbazoles, anthraquinones,phenanthridines, phenazines, azidobenzenes, psoralens, porphyrins,cholesterols, and other “conjugate” groups.

[0083] Methods of synthesizing such modified nucleosides are set forthin copending applications for United States Letters Patent, assigned tothe assignee of this invention, and entitled Compositions and Methodsfor Modulating RNA Activity, Ser. No. 463,358, filed Jan. 11, 1990;Sugar Modified Oligonucleotides That Detect And Modulate GeneExpression, Ser. No. 566,977, filed Aug. 13, 1990; and Compositions andMethods for Modulating RNA Activity, Ser. No. US91/00243, filed Jan. 11,1991, the entire disclosures of which are incorporated herein byreference.

[0084] The chirally pure phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate oligonucleotides of the invention canbe evaluated for their ability to act as inhibitors of RNA translationin vivo. Various therapeutic areas can be targeted for such antisensepotential. These therapeutic areas include but are not limited to herpesvirus (HSV), the TAR and tat regions of HIV, the codon regions ofCandida albicans chitin synthetase and Candida albicans β tubulin,papilloma virus (HPV), the ras oncogene and proto-oncogene, ICAM-1(intercellular adhesion molecule-1) cytokine, and 5′-lipoxygenase. Atargeted region for HSV includes GTC CGC GTC CAT GTC GGC. A targetedregion for HIV includes GCT CCC AGG CTC AGA TCT. A targeted region forCandida albicans includes TGT CGA TAA TAT TAC CA. A targeted region forhuman papillomavirus, e.g. virus types HPV-11 and HPV-18, includes TTGCTT CCA TCT TCC TCG TC. A targeted region for ras includes TCC GTC ATCGCT CCT CAG GG. A targeted region for ICAM-1 includes TGG GAG CCA TAGCGA GGC and the sequence CGA CTA TGC AAG TAC is a useful target sequencefor 5-lipoxygenase. In each of the above sequences the individualnucleotide units of the aligonucleotides are listed in a 5′ to 3′ sensefrom left to right.

[0085] The phosphorothioate, methylphosphonate, phosphotriester orphosphoramidate oligonucleotides of the invention may be used intherapeutics, as diagnostics, and for research, as specified in thefollowing copending applications for United States Letters Patentassigned to the assignee of this invention: Compositions and Methods forModulating RNA Activity, Ser. No. 463,358, filed Jan. 11, 1990;Compositions and Methods for Detecting and Modulating RNA Activity, Ser.No. 463,358, filed Jan. 11, 1990; Antisense oligonucleotide Inhibitorsof Papilloma Virus, Ser. No. 445,196 Filed Dec. 4, 1989; OligonucleotideTherapies for Modulating the Effects of Herpesvirus, Ser. No. 485,297,filed Feb. 26, 1990; Reagents and Methods for Modulating Gene ExpressionThrough RNA Mimicry Ser. No. 497,090, filed Mar. 21, 1990;Oligonucleotide Modulation of Lipid Metabolism, Ser. No. 516,969, filedApr. 30, 1990; Oligonucleotides for Modulating the Effects ofCytomegalovirus Infections, Ser. No. 568,366, filed Aug. 16, 1990;Antisense Inhibitors of the Human Immunodeficiency Virus, Ser. No.521,907, filed May 11, 1990; Nuclease Resistant Pyrimidine ModifiedOligonucleotides for Modulation of Gene Expression, Ser. No.558,806,filed Jul. 27, 1990; Novel Polyamine Conjugated Oligonucleotides, Ser.No. 558,663, filed Jul. 27, 1990; Modulation of Gene Expression ThroughInterference with RNA Secondary Structure, Ser. No. 518,929, filed May4, 1990; Oligonucleotide Modulation of Cell Adhesion, Ser. No. 567,286,filed Aug. 14, 1990; Inhibition of Influenza Viruses, Ser. No. 567,287,filed Aug. 14, 1990; Inhibition of Candida, Ser. No. 568,672, filed Aug.16, 1990; and Antisense Oligonucleotide Inhibitors of Papillomavirus,Ser. No. PCT/US90/07067, filed Dec. 3, 1990. These patents disclose anumber of means whereby improved modulation of RNA and DNA activity maybe accomplished through oligonucleotide interaction. To the extent thatthe specific sequences disclosed therein may be used in conjunction withthe present invention, the disclosures of the foregoing U.S. patentapplications are incorporated herein by reference.

[0086] The oligonucleotides of the invention preferably are prepared viathe process shown in Scheme 1, wherein a selected nucleotide is coupledto another nucleotide or to a growing oligonucleotide chain via anucleophilic displacement reaction. As will be recognized, this processis also applicable to the preparation of oligonucleotides comprisingnon-chiral phosphodiester linkages. In Scheme 1, R_(F) is a phosphateblocking group, B_(X) is a suitable heterocyclic nucleoside base(blocked or unblocked), R_(X) is a sugar derivatizing group and Q is anoxygen or methylene group. Together R_(O) and R_(E) are the necessaryoxygen, sulfur, nitrogen, substituted nitrogen, alkoxy or thioalkoxygroups that form the phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate linking groups. For Scheme 1, the Ygroup of the above formulas is depicted as a CPG (Controlled Pore Glass)group. Other Y groups also may be used.

[0087] In Scheme 1, a first nucleotide (13) is attached to a solid stateCPG support via its 3′-hydroxyl group in a standard manner, yieldingcompound (14). Compound (14), which forms a first synthon, is treatedwith an appropriate base, producing anionic compound (15). Compound (15)is reacted with a first unit of compound (2)—a xylofuranosyl nucleotidebearing blocking group R_(F) on its phosphate functionality and leavinggroup L at its 3′ position. Compound (2) is a second synthon. The B_(X)moiety of structure (2) may be the same as or different than the B_(X)moiety of compound (15), depending on the sequence of the desiredoligonucleotide. The anion of compound (15) displaces the leaving groupat the 3′ position of compound (2). The displacement proceeds viainversion at the 3′ position of the second synthon, forming compound(16) with n=1.

[0088] The resulting phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate linkage of compound (16) extends fromthe 5′ position of the first synthon (compound (14)) to the 3′ positionof the second synthon (compound (2)). The inversion at the 3′ positionof the second synthon results in the final configuration at the 3′nucleotide derived from the second synthon being a normal ribofuranosylsugar conformation. Compound (16) on the solid state CPG support is nowwashed to free it of any unreacted compound (2).

[0089] The second synthon carries a phosphate blocking group R_(F) onits phosphorothioate, methylphosphonate, phosphotriester orphosphoramidate phosphorus group. After coupling of the second synthonto the first synthon to yield compound (16) wherein n=1 and washing, thephosphate blocking group R_(F) is removed with an acid, yieldingcompound (17) wherein n=1. Compound (17), which represents a new firstsynthon, is now treated with base to generate a further anionic,compound (18) with n=1. Compound (18) is suitable for nucleophilicattack on a further unit of compound (2) (the second synthon) to form anew compound (16) wherein n=2. In this further unit having compound (2),the B_(X) moiety may be the same or different from the B_(X) moiety ofeither of the nucleotides of compound (16) wherein n=1, depending on thedesired sequence.

[0090] Compound (16) wherein n=2 is washed and then treated with acid todeblock the RF blocking group, yielding a further new first synthon,compound (17) wherein n=2. This new first synthon, is now ready to befurther cycled by treatment with base to yield compound (18) whereinn=2, which is now reacted with a further unit having compound (2) toyield a further unit of having structure (16) wherein n=3. Again, B_(X)may be the same or different than previously B_(X) moieties. The cycleis repeated for as many times as necessary to introduced furthernucleotides of the desired sequence via compound (2).

[0091] If it is desired to have the 5′ terminal end of the finaloligonucleotide as a phosphate group, then the last compound (17) isappropriately removed from the CPG support. If it is desired to have the5′ terminal end as a hydroxyl group, then the penultimate nucleotide isadded as compound (22), it is converted to compounds (16), (17), and(18) and the resulting compound (18) is reacted with a xylofuranosylnucleoside, compound (19). Compound (19), like compound (2), includes aleaving group L within its structure. Reaction of compound (19) withcompound (18) yields oligonucleotide, compound (20), which is releasedfrom the CPG support with concentrated ammonium ion to yield the desiredoligonucleotide, compound (21). The ammonium ion treatment will alsoremove any base blocking groups as is standard in automatedoligonucleotide synthesis.

[0092] In summary, as shown in Scheme 1, a phosphorothioate,methylphosphonate, phosphotriester or phosphoramidate 5′ nucleotide (orthe 5′-terminal nucleotide of a growing oligonucleotide) functions as afirst synthon. This is converted to an anion with a base. This aniondisplaces a leaving group at the 3′ position of a xylofuranosylnucleotide. The xylofuranosyl nucleotide comprises a second synthon. Thedisplacement proceeds via inversion at the 3′ position of the secondsynthon with the resulting phosphorothioate, methylphosphonate,phosphotriester or phosphoramidate linkage that is formed extending fromthe 5′ position of the first synthon to the 3′ position of the secondsynthon. The inversion at the 3′ position of the second synthon resultsin the final configuration at the 3′ nucleotide derived from the secondsynthon being a normal ribofuranosyl sugar conformation. It has a 3′ to4′ trans orientations (a ribofuranosyl sugar conformation) that isidentical to natural or wild type oligonucleotides.

[0093] The second synthon carries a phosphate blocking group on itsphosphorothioate, methylphosphonate, phosphotriester or phosphoramidatephosphorus group. After coupling of the second synthon to the firstsynthon, this phosphate blocking group is removed, generating a newfirst synthon having an anion at its 5′ phosphate suitable fornucleophilic attack on a further second synthon. Thus, after coupling ofthe first and second synthon, the newly joined first and second synthonsnow form a new first synthon. The oligonucleotide is elongatednucleotide by nucleotide via the nucleophilic attack of a phosphateanion at the 5′ end of the growing oligonucleotide chain on the leavinggroup at the 3′ position of the soon-to-be-added xylofuranosylconfigured second synthon nucleotide.

[0094] It is presently preferred that the phosphate blocking group be abase stable, acid labile group. Such a phosphate blocking groupmaintains the phosphate moiety of the second synthon in a protected formthat cannot react with the leaving group of the second synthon. Thisinhibits polymerization of the second synthon during the couplingreaction.

[0095] The nucleophilic coupling of the first and second synthons is astereoselective coupling process that maintains the stereospecificconfiguration about the phosphorus atom of the first synthon. Thus theparticular Sp or Rp diastereomeric configuration of a resolvedphosphorothioate, methylphosphonate, phosphotriester or phosphoramidatemoiety at the 5′ end of a starting second synthon nucleotide or the 5′terminal end of a growing first synthon oligonucleotide is maintained.While the sugar portion of the second synthon undergoes inversion aboutits 3′ position as a result of the coupling process, the phosphateportion of the second synthon retains its stereospecific configuration.After coupling of the second synthon to the first, the phosphate moiety(for example, the phosphorothioate, alkylphosphate, phosphoamidate orphosphotriester moiety) of the second synthon retains its originalstereochemistry. That is, an Rp diastereomeric second synthon retainsthe Rp configuration of its phosphate moiety, while an Sp diastereomericsecond synthon retains the Sp configuration of its phosphate moiety.

[0096] For example, to form an Rp chiral phosphorothioate,methylphosphonate, phosphotriester or phosphoramidate oligonucleotide afirst Rp diastereomeric nucleotide is chosen as the first synthon. It iscoupled with an Rp diastereomeric second synthon nucleotide. Theresulting dinucleotide maintains the Rp orientation at theinter-nucleotide linkage between the first and second nucleotides. Whena third nucleotide is next coupled to the first two, the Rpdiastereomeric phosphate moiety from the second nucleotide forms theinter-nucleotide linkage between the second and third nucleotides and ismaintained in the Rp orientation. If the third nucleotide was also an Rpdiastereomeric nucleotide then when a fourth nucleotide is added to thegrowing oligonucleotide chain, the inter-nucleotide linkage betweennucleotides three and four is also an Rp diastereomeric linkage. If eachadded “second synthon” is also an Rp diastereomer, then the resultingoligonucleotide will contain only Rp inter-nucleotide linkages. If anoligonucleotide having Sp inter-nucleotide linkages is desired, then thefirst nucleotide and each of the added subsequent nucleotides areselected as Sp diastereomeric nucleotides.

[0097] The first synthon can be a first nucleotide or a growingoligonucleotide chain. If it is desired that each of the nucleotides ofthe oligonucleotide be ribofuranoside configured nucleotides, then thefirst nucleotide is selected as a ribofuranoside configured nucleotide.Each added second synthon, while added as a xylofuranoside configurednucleotide, after inversion is converted to a ribofuranoside configurednucleotide.

[0098] The 3′ position of the first nucleotide is either blocked if asolution reaction is practiced or is coupled to a solid state support ifa solid state reaction (as for instance one utilizing a DNA synthesizer)is practiced. Each additional nucleotide of the oligonucleotide is thenderived from a xylofuranosyl nucleotide, i.e. a second synthon. Becausethe first nucleotide of the oligonucleotide can be a “standard”ribofuranosyl nucleotide coupled via its 3′ hydroxyl to a solid statesupport, the standard solid state supports known in the art, such ascontrolled pore glass (CPG) supports, can be utilized and the secondsynthons added as reagents to the growing oligonucleotide on a standardDNA synthesizer such as, for example, an Applied Biosystems Inc. 380BNucleic Acid Synthesizer. However, unlike standard DNA synthesizertechniques, nucleotide coupling is not achieved using activatedphosphoamidate chemistry. Instead, the above-noted nucleophilicdisplacement reaction of a phosphate anion on a 3′ leaving group of axylofuranosyl nucleotide is utilized as the coupling reaction. Even whenchiral phosphoramidate phosphate linkages are being incorporated intothe sequence-specific chiral oligonucleotides of the invention, thephosphoramidate groups of individual nucleotides are not directly usedto effect the coupling reaction between nucleotides. Rather, as withphosphorothioates, alkylphosphonates, and phosphotriesters, an anion isgenerated at the phosphate moiety. It is this anion—not an activatedamidate species—that is the activated species for effecting coupling.

[0099] Once the first nucleotide is loaded on a solid state supportutilizing standard techniques, the anion necessary for nucleophilicattack is generated via treatment of the first nucleotide, i.e. thefirst synthon, with a base. Suitable bases include but are not limitedto sodium hydride, methylmagnesium chloride, and t-butylmagnesiumchloride. These are used in a suitable solvent such as acetonitrile,tetrahydrofuran or dioxane.

[0100] The second synthon is added either concurrently with the base orsubsequent to it. After coupling, the growing oligonucleotide is washedwith a solvent and then treated with a reagent to effect deblocking ofthe phosphate blocking group of the second synthon. If a preferredacid-labile blocking group is used to block the phosphate of the secondsynthon, deblocking is easily effected by treating the growingoligonucleotide on the solid state support with an appropriate acid.

[0101] Suitable acid-labile blocking groups for the phosphates of thesecond synthon include but are not limited to t-butyl, dimethoxytrityl(DMT) or tetrahydropyranyl groups. Suitable acids for deblocking thesecond synthon phosphate blocking group include but are not limited toacetic acid, trichloroacetic acid, and trifluoromethane sulfonic acid.Such acids are suitably soluble in solvents such as tetrahydrofuran,acetonitrile, dioxane, and the like.

[0102] Following treatment with an appropriate deblocking reagent toeffect deblocking of the phosphate protecting group, the growingoligonucleotide is then washed with an appropriate solvent such astetrahydrofuran, acetonitrile or dioxane. The oligonucleotide is nowready for the addition of a further nucleotide via treatment with baseto generate an anion on the 5′ terminal phosphate followed by theaddition of a further second synthon. Alternatively, the anion can begenerated concurrently with addition of a further second synthon.Suitable leaving groups for inclusion at the 3′ position of thexylofuranosyl second synthon include but are not limited to the groupconsisting of halogen, alkylsulfonyl, substituted alkylsulfonyl,arylsulfonyl, substituted arylsulfonyl, hetercyclcosulfonyl ortrichloroacetimidate. A more preferred group includes chloro, fluoro,bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl,methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate),p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate),trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl,imidazolesulfonyl, and 2,4,6-trichlorophenyl. These leaving groups aresubject to SN₂ displacement reactions with inversion about the 3′position of the sugar to provide the required 3′-4′ trans ribofuranosylconfiguration after inversion. The ionized oxygen atom of the phosphatemoiety of the first synthon displaces these leaving groups to effect thecoupling of the first synthon to the second synthon.

[0103] A further class of leaving groups include certain sugar-basecyclonucleosides. These include 2,3′ or 6,3′-cyclopyrimidines and8,3′-cyclopurine nucleosides. These nucleosides are alternately known asanhydro nucleosides. Since the sugar-heterocycle bond of suchcyclonucleosides is “syn” with the heterocycle base, nucleophilicaddition with inversion at this site also yields the desiredribofuranoside configuration of the added nucleotide after addition ofthe second synthon to the first synthon. The linking atom between the 3′position of the sugar and the 2 or 6 position of a pyrimidine base orthe 3′ position of the sugar and the 8 position of the purine base canbe oxygen, sulfur or nitrogen.

[0104] Since a basic environment is created during coupling of the firstsynthon to the second synthon and an acidic environment (utilizing thepreferred acid-labile phosphate blocking group) is created duringdeblocking of the phosphate blocking group from the nucleotide derivedfrom the second synthon, if blocking groups are utilized on the base orsugar portions of the nucleotides such base or sugar blocking groupsmust be stable to both acidic and basis conditions. Suitable blockinggroups for the heterocyclic base or the sugar are selected to be stableto these conditions. One type of blocking groups that can be used areacid\base stable, hydrogenolysis-sensitive blocking groups; that is,blocking groups which can be removed with molecular hydrogen but notwith acid or base. A benzyl blocking group is such a suitablehydrogenolysis-sensitive blocking group.

[0105] Other heterocycle base or sugar blocking groups are those thatrequire more pronounced acid or base treatment to de-block than may beexperienced during the basic activation of the nucleophilic displacementreaction of the second synthon blocking group or the acidic removal ofthe phosphate blocking group. Two such blocking groups are the benzoyland isobutyryl groups. Both of these require strong basic conditions fortheir removal. These basic conditions are more stringent than thatrequired to generate the phosphate anion for the nucleophilicdisplacement reaction. This allows the use of such benzoyl or isobutyrylblocking groups for the 6-amino group of adenine, the 2-amino group ofguanine, and the 4-amino group of cytosine. Suitable precursor moleculesfor the second synthons include the xylo derivatives of the commonnucleosides. Certain of these “xylo nucleosides” are commerciallyavailable and others are known in the nucleoside literature.

[0106] Xylo nucleosides include but are not limited to xylo derivativesof adenosine, guanosine, inosine, uridine, cytidine, thymidine,5-methylcytidine, and 2-aminoadenosine, i.e.9-(β-D-xylofuranosyl)adenine, 9-(β-D-xylofuranosyl)guanine,9-(β-D-xylofuranosyl)hypoxanthine, 1-(β-D-xylofuranosyl)uracil,1-(β-D-xylofuranosyl)cytosine, 1-(β-D-xylofuranosyl)thymine,5-methyl-1-(β-D-xylofuranosyl)cytosine, and2-amino-9-(β-D-xylofuranosyl)adenine. They also include the xyloequivalents of the common 2′-deoxy nucleosides such as9-(β-D-2′-deoxy-threo-pentofuranosyl)adenine,9-(β-D-2′-deoxy-threo-pentoufuranosyl)guanine,9-(β-D-2′-deoxy-threo-pentofuranosyl)hypoxanthine,1-(β-D-2′-deoxy-threo-pentofuranosyl)uracil,1-(β-D-2′-deoxy-threo-pentofuranosyl)cytosine,1-(β-D-2′-deoxy-threo-pentofuranosyl)thymine,5-methyl-1-(β-D-2′-deoxy-threo-pento-furanosyl)cytosine, and2-amino-9-(β-D-2′-deoxy-threo-pento-furanosyl)adenine.

[0107] Other preferred nucleosides that are suitable precursors for thesecond synthon include but are not limited to 2′-fluoro, 2′-methoxy,2′-O-allyl, 2′-methyl, 2′-ethyl, 2′-propyl, 2′-chloro, 2′-iodo,2′-bromo, 2′-amino, 2′-azido, 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-nonyl, 2′-O-pentyl, 2′-O-benzyl, 2′-O-butyl,2′-O-(propylphthalimide), 2′-S-methyl, 2′-S-ethyl, 2′-aminononyl,2′-aralkyl, and 2′-alkylheterocyclo such as propylimidazoyl derivativesof the above 2′-deoxy-threo-pentofuranosyl nucleosides. Representativesof this group include but are not limited to9-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)adenine,9-(β-D-2′-deoxy-2′-fluoro-threo-penofuranosyl)guanine,9-(β-D-2′-deoxy-2-fluoro-threo-pentofuranosyl)hypoxanthine,1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)uracil,1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)cytosine,1-(β-D-2′-deoxy-2′-fluoro-threo-prntofuranosyl)thymine,5-methyl-1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)cytosine,2-amino-9-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)adenine,9-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl)adenine,9-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl)guanine,9-(β-D-2′-deoxy-2-methoxy-threo-pentofuranosyl)hypoxanthine,1-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl)uracil,1-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl)cytosine,1-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl)thymine,5-methyl-1-(β-D-2′-deoxy-02′-methoxy-threo-pentofuranosyl)cytosine,2-amino-9-(β-D-2′-deoxy-2′-methoxy-threo-pentofuranosyl)adenine,9-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl)adenine,9-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl)guanine,9-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl)hypoxanthine,1-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl)uracil,1-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl)cytosine,1-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl)thymine,5-methyl-1-(β-D-2′-deoxy-2′-O-allyl-threo-pentofuranosyl)cytosine,2-amino-9-(β-D-2′-O-allyl-threo-pentofuranosyl)adenine,9-(β-D-2′-deoxy-2′-methyl-threo-pentofuranosyl)adenine,9-(β-D-2′-deoxy-2′-chloro-threo-pentofuranosyl)guanine,9-(β-D-2′-deoxy-2-amino-threo-pentofuranosyl)hypoxanthine,1-(β-D-2′-deoxy-2′-O-nonyl-threo-pentofuranosyl)uracil,1-(β-D-2′-deoxy-2′-O-benzyl-threo-pentofuranosyl)cytosine,1-(β-D-2′-deoxy-2′-bromo-threo-pentofuranosyl)thymine,5-methyl-1(βD-2′-deoxy-2′-O-butyl-threo-pentofuranosyl)cytosine, and2-amino-9-[β-D-2′-deoxy-2′-O-(propylphthalimide)-threo-pentofur-anosyl)adenine.The 2′-deoxy-2′-fluoro-threo-pentofuranosyl, and2′-deoxy-2′-methoxy-threo-pentofuranosyl nucleosides are particularlypreferred in that the 2′-fluoro and 2′-methoxy groups give improvednuclease resistance to oligonucleotide bearing these substituents ontheir respective nucleotides.

[0108] Further preferred nucleosides that are suitable precursors forthe second synthon include but are not limited to the xylofuranosyl or2′-deoxy-threo-pentofuranosyl derivatives of 3-deaza purine and2-substituted amino purine nucleosides including but not limited to3-deaza-2′-deoxyguanosine, 3-deaza-3-nonyl-2′-deoxyguanosine,3-deaza-3-allyl-2′-deoxyguanosine, 3-deaza-3-benzyl-2′-deoxyguanosine,3-deaza-3-nonyl-2′-deoxyguanosine,N2-[imidazol-1-yl-(propyl)]-2′-deoxy-guanosine, and2-amino-N2-[imidazol-1-yl(propyl)]adenosine.

[0109] Another preferred group of nucleoside precursors for the secondsynthon include the carbocyclic nucleosides, i.e. nucleosides having amethylene group in place of the pentofuranosyl ring oxygen atom. Suchcarbocyclic compounds may exhibit increased stability towards chemicalmanipulation during activation of the xylo nucleosides for nucleophilicattack.

[0110] The xylo nucleoside or derivatized xylo nucleoside is reactedwith a suitable phosphorylating agent to phosphorylate the secondsynthon precursor. Various phosphorylation reactions are known in theart such as those described in Nucleotide Analogs, by Karl Heinz Scheit,John Wiley & Sons, 1980, Chapter Four—Nucleotides with ModifiedPhosphate Groups and Chapter Six—Methods Of Phosphorylation; ConjugatesOf Oligonucleotides and Modified Oligonucleotides: A Review Of TheirSynthesis and Properties, Goodchild, J. (1990), Bioconjugate Chemistry,1:165; and Antisense Oligonucleotides: A New Therapeutic Principle,Uhlmann, E. and Peyman, A. (1990), Chemical Reviews, 90:543.

[0111] Preferred phosphorylating agents include phosphoryl chlorides.Suitable phosphoryl chlorides include but are not limited tothiophosphoryl chloride, t-butoxyphosphoryl chloride,t-butoxy(methyl)phosphoryl chloride, t-butoxy-(methyl)thiophosphorylchloride, t-butoxy(methoxy)phosphoryl chloride. Other phosphorylchlorides nay include t-butoxy(N-morpholino)phosphoryl chloride,t-butoxy(ethoxy-ethylamino)phosphoryl chloride,t-butoxy(methy-thioxy)phosphoryl chloride, and the like. Suchphosphorylating agents are utilized to yield the correspondingphosphorothioate, phosphoramidate, phosphotriester, alkylphosphonates,and phosphodiester xylo nucleotides.

[0112] Even enzymatic phosphorylation is possible, as for example thephosphorylation of 9-(β-D-xylofuranosyl)guanine by nucleosidephosphotransferase from Pseudomonas trifolii as per the procedure ofSuzaki, S., Yamazaki, A. Kamimura, A., Mitsugi, K., and Kumashior, I.(1970), Chem. Pharm. Bull. (Tokyo), 18:172.

[0113] 1-(β-D-Xylofuranosyl)uracil 5′-phosphate was identified but notseparated from its 3′ isomer as reported by Holy, A. and Sorm, F.(1969), Coll. Czech. Chem. Commun., 34:1929. Also,9-(2′-O-benzyl-β-D-xylofuranosyl)adenine 5′-phosphate was obtained as anintermediate by Hubert-Habart, M. and Goodman, L. (1969), Chem. Commun.,740. Removable of the benzyl blocking group would give the desiredunblocked nucleotide.

[0114] Additionally, the alkylphosphonates can be prepared by the methodof Holy, A. (1967), Coll. Czech. Chem. Commun., 32:3713.Phosphorothioates have also been prepared by treatment of thecorresponding nucleoside with trisimidazolyl-1-phosphinesulfide followedby acid hydrolysis with aqueous acetic acid, Eckstein, F. (1966 & 1970),J. Am. Chem. Soc., 88:4292 & 92:4718, respectively. A more preferredmethod is by selective thiophosphorylation by thiophosphorylchloride intriethylphosphate, Murray, A. W. and Atkinson, M. R. (1968),Biochemistry, 7:4023.

[0115] The appropriate phosphorylated xylo nucleotide is then activatedfor nucleophilic displacement at its 3′ position by reacting the3′-hydroxyl group of the xylo compound with an appropriate anhydride,chloride, bromide, acyloxonium ion, or through an anhydro or cyclonucleoside or the like to convert the 3′-hydroxyl group of the xylonucleoside to an appropriate leaving group.

[0116] In a further method of synthesis, treatment of2′,3′-anhydroadenosine with sodium ethylmercaptide gives9-[3-deoxy-3-(ethylthio)-β-D-xylofuranosyl]adenine. Treatment of thiscompound with a first synthon nucleophile may generate a terminal2-ethylthio arabinofuranosyl nucleoside that could be desulfurized toyield the corresponding 2′-deoxynucleoside.

[0117] If during phosphorylation or conversion of the xylo 3′-hydroxylto a 3′-activated leaving group stereospecific diastereomers are notobtained, after completion of the phosphorylation or conversion of the3′-hydroxyl to an activated leaving group, the Rp and Sp diastereomersof these compounds will then be isolated by HPLC. This will yield purediastereomers in a stereospecific form ready for use as the secondsynthons of Scheme 1.

[0118] Additional objects, advantages, and novel features of thisinvention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are not intended tobe limiting.

EXAMPLE 1 Xyloadenosine Method A—Condensation Reaction

[0119] Adenine is condensed with1,2,3,5-tetra-O-acetyl-D-xylopentofuranoside in the presence of TiCl₄ asthe condensation catalyst in a polar solvent utilizing the method ofSzarek, W. A., Ritchie, R. G. S., and Vyas, D. M. (1978), Carbohydr.Res., 62:89.

Method B—Alkylation Reaction

[0120] 8-Mercaptoadenine is alkylatedwith5-deoxy-5-iodo-1,2-O-isopropylidine-xylofuranose followed bytreatment with acetic acid/acetic anhydride/sulfuric acid and thenammonia. This yields an 8-5′-anhydro intermediate nucleoside that isoxidized with aqueous N-bromosuccinimide to give the sulfoxide. This isblocked with benzoic anhydride and after a Pummerer rearrangement can bedesulfurized with Rainey nickel to give 9-β-D-xylofuranosyladenine asper the procedure of Mizuno, Y., Knaeko, C., Oikawa, Y., Ikeda, T, andItoh, T. (1972), J. Am. Chem. Soc., 94:4737.

EXAMPLE 2 3-Deaza-9-(β-D-xylofuranosyl)guanine

[0121] In a manner similar to Example 1, Method A, 3-deazaguanine iscondensed with 1,2,3,5-tetra-O-acetyl-D-xylopentofuranoside to yield thetitle compound.

EXAMPLE 3 N⁶-Benzoyl-9-(2′-Deoxy-2′-fluoro-threo-pentofuranosyl)adenine

[0122] In a manner similar to Example 1, Method A, N⁵-benzoyladenine iscondensed with1,3,5-tri-O-acetyl-2-deoxy-2-fluoro-D-threo-pentofuranoside to yield thetitle compound.

EXAMPLE 4 1-(2′-Deoxy-2′-methoxy-β-D-xylofuranosyl)uridine

[0123] In a manner similar to Example 1, Method A, uracil is condensedwith 1,3,5-tri-O-acetyl-2-deoxy-2-methoxy-D-threo-pentofuranoside toyield the title compound.

EXAMPLE 5 1-(2′-Deoxy-2′-O-allyl-β-D-threo-pentofuranosyl)cytosine

[0124] In a manner similar to Example 1, Method A, cytosine is condensedwith 1,3,5-tri-O-acetyl-2-deoxy-2-O-allyl-D-threo-pentofuranoside toyield the title compound.

EXAMPLE 6 Xyloguanosine Method A

[0125] In a manner similar to Example 1, Method A, guanine is condensedwith 1,2,3,5-tetra-O-acetyl-D-xylopentofuranoside to yield the titlecompound.

Method B

[0126] The chloromercury derivative of 2-acetamido-6-chloropurine iscondensed with 2,3,5-tri-O-acetyl-B-D-ribofuranosylpurine utilizing themethod of Lee et. al. (1971), J. Med. Chem., 14:819. The condensationproduct was treated with ammonia to yield2-amino-6-chloro-9-(β-D-xylofuranosyl)purine. Further treatment withsodium hydroxyethylmercaptide gives the title compound.

EXAMPLE 7 2-Amino-6-mercapto-9-(β-D-xylofuranosyl)purine

[0127] 2-Amino-6-chloro-9-(β-D-xylofuranosyl)purine as prepared by theExample 6, Method B, is treated with sodium hydrosulfide to give thetitle compound.

EXAMPLE 8 9-(2′-Deoxy-2′-methyl-β-D-threo-pentofuranosyl)guanine

[0128] In a manner similar to Example 1, Method A, guanine is condensedwith 1,3,5-tri-O-acetyl-2-deoxy-2-methyl-D-threo-pentofuranoside toyield the title compound.

EXAMPLE 9 2-Amino-xyloadenosine

[0129] 2-amino-8-mercaptoadenine is treated in the manner as per Example6, Method B to yield the title compound.

EXAMPLE 10 Carbocyclic Xyloadenosine

[0130] 5-Amino-4,6-dichloropyrimidine is treated with(±)-4α-amino-2α,3β-dihydroxy-1α-cyclopentanemethanol to give apyrimidine intermediate that is aminated and ring closed to yield thecarbocyclic analog of xylofuranosyladenine as per the procedure ofVince, R. and Daluge, S. (1972), J. Med. Chem., 15:171.

EXAMPLE 11 Carbocyclic Xyloinosine

[0131] 5-Amino-6-chloro-pyrimidyl-4-one when treated with(±)-4α-amino-2α,3β-dihydroxy-1α-cyclopentanemethanol will give apyrimidine intermediate that is then aminated and ring closed to yieldthe carbocyclic analog of xylofuranosylinosine as per the procedure ofExample 8.

EXAMPLE 12 O²,3′-Cyclothymidine Method A

[0132] 3′-O-Mesylthymidine is treated with boiling water and the pH isadjusted to pH 4-5 according to the procedure of Miller, N. and Fox, J.J. (1964), J. Org. Chem., 29:1771 to yield the title compound. This samecompound can also prepared from 3′-deoxy-3′-iodothymidine by treatmentwith silver acetate in acetonitrile.

Method B

[0133] O²,3′-Cyclothymidine and other 2′-deoxynucleosides are preparedby the treatment of the appropriate nucleoside with(2-chloro-1,1,3-trifluoroethyl)diethylamine in dimethylformamideaccording to the procedure of Kowollik, G., Gaertner, K., and Langen, P.(1969), Tetrahedron Lett., 3863.

EXAMPLE 13 O²,3′-Cyclouridine

[0134] 3′-O-tosyluridine is treated with t-butoxide according to theprocedure of Letters, R. and Michelson, A. M. (1961), J. Chem. Soc.,1410. This compound is also prepared by treatment of3′-O-mesyl-2′,5′-di-O-trityluridine with sodium benzoate indimethylformamide followed by detritlation with hydrochloric inchloroform.

EXAMPLE 14 S²,3′-Cyclo-2-thiothymidine Method A

[0135] 3′-O-mesyl-O²,5′-cyclothymidine is subjected to methanolysisfollowed by sulfhydryl ion attack. The S²,3′-cyclo linkage is thenopened up with base to yield2′,3′-dideoxy-3′-mercapto-1-(β-D-xylofuranosyl)thymidine as per theprocedure of Wempen, I. and Fox, J. J. (1969), J. Org. Chem., 34:1020.

Method B

[0136] S²,3′-Cyclo-2-thiouridine is also prepared from 2-thiouridine bythe method of Doerr, I. L. and Fox, J. J. (1967), J. Am. Chem., 89:1760.

EXAMPLE 15 N⁶,5′-Cyclothymidine

[0137] 5′-O-Trityl-3′-O-mesylthymidine is treated with sodium azide toyield N⁶,5′-cyclothymidine as one of the products.5′-O-trityl-3′-O-mesylthymidine is also cyclizable toO²,3′-cyclothymidine.

EXAMPLE 16 8,3′-Cycloadenosine

[0138] The anhydro ring from the 3′ position of the sugar to the 8position of the purine ring is formed by treatment of5′-O-acetyl-8-bromo-2′ (or 3′)-O-p-toluenesulfonyladenosine withthiourea to yield the 8,3′-thiocyclonucleoside (as well as thecorresponding 8,2′) product as per the procedure of Ikehara, M. andKaneko, M. (1970), Tetrahedron, 26:4251.

EXAMPLE 17 8,3′-Cycloguanosine

[0139] The title compound is prepared as per Example 16 utilizing8-bromoguanosine. Both this compound and the compound of Example 16 canbe oxidized to their corresponding sulfoxides via tert-butylhypochlorite in methanol or treated with chlorine in methanolic hydrogenchloride to yield the 3′-sulfo-8-chloro analog in a procedure analogouswith that of Mizuno, Y., Kaneko, O., and Oikawa, Y. (1974), J. Org.Chem., 39:1440.

EXAMPLE 181-(3′-Bromo-3′-deoxy-N⁴,2′,5′-O-triacetylxylofuranosyl)cytosine

[0140] The title compound is prepared by treating N⁴-acetylcytidine withacetyl bromide according to the procedure of Karumoto, R. and Honjo, M.(1974), Chem. Pharm. Bull., 22:128.

EXAMPLE 19 9-(3-Deoxy-3-fluoro-β-D-xylofuranosyl)adenine

[0141] Treatment of9-(2,3-anhydro-5-O-benzoyl-β-D-ribofuranosyl)-N,N-dibenzoyl (orN-pivaloyl)adenine with tetraethylammonium fluoride in hot acetonitrilefollowed by deacylation with sodium methoxide yields9-(3-deoxy-3-fluoro-β-D-xylofuranosyl)adenine as per the procedure ofLichtenthaler, F. W., Emig, P., and Bommer, D. (1969), Chem. Ber.,102:964.

EXAMPLE 20 9-(3-Deoxy-3-fluoro-β-D-xylofuranosyl)guanine

[0142] In a like manner to Example 18 the corresponding guanosinecompound will be prepared from the corresponding 2,3-anhydro guanosine.

EXAMPLE 21 9-(3′-Chloro-3′-deoxy-β-D-xylofuranosyl)hypoxanthine

[0143] 5′-O-Acetylinosine is treated with triphenylphosphine and carbontetrachloride to yield the title compound according to the procedure ofHaga, K., Yoshikawa, M., and Kato, T. (1970), Bull. Chem. Soc. Jpn.,43:3992.

EXAMPLE 229-(2-O-Acetyl-3-chloro-3-deoxy-5-O-pivaloyl-β-D-xylofurano-syl)-6-pivalamidopurine

[0144] The title compound is prepared via an intermediate2′,3′-O-acyloxonium ion utilized to introduce a halogen atom at the 3′position and convert the ribo configuration of a nucleoside into thecorresponding 3′-halo-3′-deoxy xylo nucleoside. The acyloxonium ion isgenerated in situ by treatment of 2′,3′-O-methoxyethylidineadenosinewith pivaloyl chloride in hot pyridine. Attack by chloride gives thetitle compound. Hypoxanthine and guanine nucleoside react in a similarmanner. Sodium iodide will be used to generate the corresponding3′-iodides according to the procedure of Robins, M. J., Fouron, Y., andMengel, R. (1974), J. Org. Chem., 39:1564.

EXAMPLE 23 9-(2,5,-Di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine

[0145] This compound is prepared by a in situ acyloxonium ion generatedby treating 3′,5′-di-O-acetyladenosine with boron trifluoride etheratefollowed by phosphorus tribromide according to the procedure of Kondo,K., Adachi, T., and Inoue, I. (1977), J. Org. Chem., 42:3967. The titlecompound can also be formed by treating adenosine withtetraacet-oxysilane and phosphorus tribromide in the presence of borontrifluoride etherate.

EXAMPLE 24 1-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)uracil

[0146] In a manner similar to Example 3, uracil is condensed with1,3,5,-tri-O-acetyl-2-deoxy-2-fluoro-D-threo-pentofuranoside to yieldthe title compound.

EXAMPLE 25 1-(β-D-2′-Deoxy-2′-fluoro-threa-pentofuranosyl)guanine

[0147] In a manner similar to Example 3, guanine is condensed with1,3,5,-tri-O-acetyl-2-deoxy-2-fluoro-D-threo-pentofuranoside to yieldthe title compound.

EXAMPLE 26 1-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)cytosine

[0148] In a manner similar to Example 3, cytosine is condensed with1,3,5,-tri-O-acetyl-2-deoxy-2-fluoro-D-threo-pentofuranoside to yieldthe title compound.

EXAMPLE 27 O²,3′-Cyclo-2′-deoxycytidine

[0149] The title compound is prepared by heating the 3′-O-sulfamate asper the procedure of Schuman, D., Robins, M. J., and Robins, R. K.(1970), J. Am. Chem. Soc., 92:3434.

EXAMPLE 28 Sp and Rp Xyloadenosine 5′-Monophosphate

[0150] N⁶-Benzoyl-xyloadenosine is phosphorylated with phosphorylchloride in pyridine and acetonitrile at 0° C. The reaction will bequenched with ice water, rendered basic and added to an activatedcharcoal column. After elution with ethanol/water/concentrated ammoniumhydroxide the solvent is evaporated to dryness and the residue dissolvedin water and passed through an ion exchange column. The benzoyl blockinggroup is removed in concentrated ammonium hydroxide followed byseparation of the diastereomers by HPLC to yield the title compound.

EXAMPLE 29 Sp and Rp1-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)uracil5′-t-butoxy(methyl)phosphonate

[0151] 1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)thymine will bephosphorylated with t-butoxy(methyl)phosphoryl chloride intrimethylphosphate at 0° C. for 3 hrs. The solution is added to coldanhydrous ether. The racemic precipitate is taken up in acetonitrile andthe Sp and Rp diastereomers of the title compound separated by HPLCutilizing a gradient of acetonitrile and triethylammonium acetatebuffer.

EXAMPLE 30 Sp and Rp1-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)cytosine5′-t-butoxy(methyl)phosphonate

[0152] 1-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)cytosine will bephosphorylated and purified as per the procedure of Example 29 to givethe diastereomers of the title compound.

EXAMPLE 31 Sp and RpN⁵-Benzoyl-9-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)adenine5′-t-butoxy(methyl)phosphonate

[0153] N⁶-Benzoyl-9-(β-D-2′-deoxy-2′-fluoro-threo-pentofuranosyl)adeninewill be phosphorylated and purified as per the procedure of Example 29to give the diastereomers of the title compound.

EXAMPLE 32 Sp and Rp9-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)guanine5′-t-butoxy(methyl)phosphonate

[0154] 9-(β-D-2′-Deoxy-2′-fluoro-threo-pentofuranosyl)guanine will bephosphorylated and purified as per the procedure of Example 29 to givethe diastereomers of the title compound.

EXAMPLE 33 Sp and Rp Xylofuranosyluracil 5′-t-butoxyphosphorothioate

[0155] Xylofuranosyluracil will be phosphorothioated witht-butoxythiophosphorylchloride in triethylphosphate utilizing the methodof Murray, A. W. and Atkinson, M. R. (1968), Biochemistry, 7:4023. Thediastereomers of the title compound are separated by HPLC.

EXAMPLE 34 Sp and Rp9-(2′-Deoxy-2′-methyl-β-D-threo-pentofuranosyl)guanine5′-Methylphosphonate

[0156] 9-(2′-Deoxy-2′-methyl-β-D-threo-pentofuranosyl)guanine will-bealkylphosphonated utilizing the procedure of Holy, A. (1967), Coll.Czech. Chem. Commun., 32:3713. The racemic phosphorylation product isseparated into its Sp and Rp diastereomers using HPLC chromatography toyield the title compound.

EXAMPLE 35 Sp and Rp 9-(2′-Deoxy-β-D-threo-pentofuranosyl)hypoxanthine5′-Phosphormorpholidate

[0157] 9-(2′-Deoxy-β-D-threo-pentofuranosyl)hypoxanthine isphosphorylated according to the procedure of Example 28. The resulting5′-phosphate intermediate will be phosphormorpholidated by treatmentwith activated with dicyclohexylcarbodiimide in the presence ofmorpholine according to the procedure of Moffatt, J. G. and Khorana, H.G. (1961), J. Am. Chem. Soc., 83:3752 to yield the racemic titlecompound. The diastereomers of the product are separated by HPLC.

EXAMPLE 36 Sp and Rp9-(2′-Deoxy-2′-O-allyl-β-D-threo-pentofuranosyl)cytosine 5′-Phosphate

[0158] 9-(2′-Deoxy-2′-allyl-β-D-threo-pentofuranosyl)cytosine will bephosphorylated according to the procedure of Example 28 to yield theracemic title compound. The diastereomers are separated by HPLC.

EXAMPLE 37 Sp and Rp9-(2′-Deoxy-2′-methoxy-β-D-threo-pentofuranosyl)-uracil 5′-Phosphate

[0159] 9-(2′-Deoxy-2′-methoxy-β-D-threo-pentofuranosyl)uracil will bephosphorylated according to the procedure of Example 28 to yield theracemic title compound. The diastereomers are separated by HPLC.

EXAMPLE 38 Sp and Rp 3-Deaza-9-(xylofuranosyl)guanine 5′-Phosphate

[0160] 3-Deaza-9-(xylofuranosyl)guanine will be phosphorylated accordingto the procedure of Example 28 to yield the racemic title compound. Thediastereomers are separated by HPLC.

EXAMPLE 39 Sp and Rp Xyloguanosine 5′-Phosphorothioate

[0161] Xyloguanosine will be phosphorothioated with thiophosphorylchloride according to the procedure of Example 28 to yield the racemictitle compound. The diastereomers are separated by HPLC.

EXAMPLE 40 Sp and Rp Carbocyclic Xyloadenosine 5′-Phosphate

[0162] In a like manner to Example 28, carbocyclic xyloadenosine will betreated with phosphoryl chloride to yield the racemic title compound.The diastereomers will be separated by HPLC.

EXAMPLE 41 Activated 3′-Deoxy-3′-Active Leaving Group PhosphorylatedNucleasides

[0163] The 3′-halo nucleotides can be treated with methoxide to give anunstable 2′,3′-anhydro intermediate that slowly forms the corresponding3,3′-cyclonucleoside. The cyclonucleoside in turn can undergonucleophilic attack to yield other 3′-deoxy-3′-substituted derivatives,as for instance, the tosyl, triflate, trichloroacetimidate or otheractive species.

EXAMPLE 42N⁶-Benzoyl-9-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl)adenine5′-Rp t-Butoxy(methyl)phosphonate

[0164] N⁶-Benzoyl-9-(2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl)adenine5′-Rp t-butoxy(methyl)phosphonate will be treated withp-toluenesulfonlychloride in pyridine as per the procedure of Reist, E.J., Bartuska, V. J., Calkins, D. F., and Goodman, L. (1965), J. Org.Chem., 30:3401 to yield the title compound.

EXAMPLE 439-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-methoxy-β-D-threo-pentofuranosyl)uracil5′-Rp t-Butoxy(methyl)phosphonate

[0165] 9-(2′-Deoxy-2′-methoxy-β-D-threo-pentofuranosyl)uridine 5′-Rpt-butoxy(methyl)phosphonate will be treated withp-toluenesulfonylchloride in pyridine according to the procedure ofExample 42 to yield the title compound.

EXAMPLE 449-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl)uracil5′-Rp t-Butoxy(methyl)phosphonate

[0166] 9-(2′-Deoxy-2′-fluoro-β-D-threo-pentofuranosyl)uridine 5′-Rpt-butoxy(methyl)phosphonate will be treated withp-toluenesulfonlychloride in pyridine according to the procedure ofExample 42 to yield the title compound.

EXAMPLE 459-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl)cytosine5′-Rp t-Butoxy(methyl)phosphonate

[0167] 9-(2′-Deoxy-2′-fluoro-β-D-threo-pentofuranosyl)cytosine 5′-Rpt-butoxy(methyl)phosphonate will be treated withp-toluenesulfonlychloride in pyridine according to the procedure ofExample 42 to yield the title compound.

EXAMPLE 469-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofurano-syl)guanine5′-Sp Phosphate

[0168] 9-(2′-Deoxy-2′-fluoro-β-D-threo-pentofuranosyl)guanine 5′-Spphosphate will be treated with p-toluenesulfonly chloride in pyridineaccording to the procedure of Example 42 to yield the title compound.

EXAMPLE 47 9-(3′-Deoxy-3′-tosyl-2′-deoxy-2′-O-allyl-β-D-threo-pentofuranosyl)thymine 5′-Rp Phosphate

[0169] 9-(2′-deoxy-2′-O-allyl-β-D-threo-pentofuranosyl)thymine 5′-Rpphosphate will be treated with p-toluenesulfonyl chloride in pyridineaccording to the procedure of Example 42 to yield the title compound.

EXAMPLE 48 3′-Deoxy-3′-trifluoromethanesulfonylxyloguanosine 5′-SpPhosphorothioate

[0170] Xyloguanosine 5′-Sp phosphorothioate will be treated withtrifluoromethane sulfonic acid anhydride in the presence of a sodiumhydride to yield the title compound.

EXAMPLE 49 Carbocyclic3′-Deoxy-3′-trifluoromethanesulfonylxyloadenosine5′-Rp Phosphate

[0171] In a like manner to Example 48, carbocyclic xyloadenosine 5′-Rpphosphate will be treated with trifluoromethane sulfonic acid to yieldthe title compound.

EXAMPLE 50 S²,3′-Cyclo-2-thiothymidine

[0172] S²,3′-Cyclo-2-thiothymidine is prepared from3′-O-mesyl-O²,5′-cyclothymidine via methanolysis followed by sulfhydrylion attack. The S²,3′-cyclo linkage is then opened up with base to yield2′,3′-dideoxy-3′-mercapto-1-(β-D-xylofuranosyl)thymidine, Wempen, I. andFox, J. J. (1969), J. Org. Chem., 34:1020. The 3′ position will then beactivated to nucleophilic attack via an active leaving group such asconversion of the mercapto to a tosyl leaving group. In a like mannerS²,3′-Cyclo-2-thiouridine prepared from 2-thio-uridine by the method ofDoerr, I. L. and Fox, J. J. (1967), J. Am. Chem., 89:1760, can be ringopened and then derivatized with an activated leaving group such as atosylate.

EXAMPLE 51 Synthesis of 2′-Deoxy-2′-fluoro substituted CGA CTA TGC AACTAC Rp Methylphosphonate Linked Oligonucleotide

[0173] 1-(2′-Fluoro-2′-deoxy-β-D-riboturanosyl)cytosine 5′-Rpmethylphosphonate will be attached via its 3′ hydroxyl to CPG beads in astandard manner as practiced in automated nucleic acid synthesis. Thisnucleotide forms the first synthon 1

[0174] (a) Activation of Synthon 1

[0175] The beads are washed with acetonitrile and treated with 1.1equivalents of sodium hydride in acetonitrile to form an anion on themethylphosphonate moiety.

[0176] (b) Addition of Synthon 2 and Coupling of Synthons 1 and 2

[0177] 2.0 Equivalents ofN⁶-Benzoyl-9-(3′-deoxy-3′-tosyl-2′-deoxy-2′-fluoro-β-D-threo-pentofuranosyl)adenine5′-Rp t-butoxy(methyl)phosphonate in acetonitrile is added withstirring. After completion of the nucleophilic reaction and formation ofthe cytosine-adenine dimer as judged by tlc, the beads are filtered andwashed with acetonitrile.

[0178] (c) Removal of t-Butoxy Blocking Group

[0179] The beads are re-suspended in acetonitrile and 1.5 equivalents oftrichloracetic acid is added. The reaction is stirred to remove thet-butoxy blocking group on the terminal 5′-methylphosphonate group ofthe adenosine nucleotide, followed by washing with acetonitrile.

[0180] (d) Cycling

[0181] The reaction is cycled to step (a), followed by addition of thenext synthon 2 nucleotide at step (b), and deblocking at step (c). Thereaction is further cycled for each nucleotide making up the specificsequence of the oligonucleotide. After the addition of the penultimatenucleotide its phosphate moiety is activated at step (a) and the finalnucleosidic unit is added at step (b) as a xylofuranosyl nucleoside. Theoligonucleotide is concurrently deblocked and removed from the CPG beadsby treatment with concentrate ammonium hydroxide.

What is claimed is:
 1. A method for preparing an oligonucleotidecomprising a predetermined sequence of nucleoside units, comprising thesteps of: (a) selecting a first synthon having structure:

(b) selecting a second synthon having structure:

(c) contacting the second synthon with the first synthon in the presenceof a base to effect nucleophilic attack of a 5′-phosphate of the firstsynthon at a 3′-position of the second synthon to yield a new firstsynthon via a stereospecific inversion of configuration at the 3′position of the second synthon; and (d) treating the new first synthonwith a reagent to remove the blocking group R_(F); wherein: Q is O orCH₂; R_(D) is O, S, methyl, alkoxy, thioalkoxy, amino or substitutedamino; R_(E) is O or S; R_(F) is H or a labile blocking group; R_(X) isH, OH, or a sugar derivatizing group; B_(X) is a naturally occurring orsynthetic nucleoside base or blocked nucleoside base; L is a leavinggroup or together L and Bx are a 2-3′ or 6-3′ pyrimidine or 8-3′ purinecyclo-nucleoside; and Y is a stable blocking group, a solid statesupport, a nucleotide on a solid state support, or an oligonucleotide ona solid state support.
 2. The method of claim 1 wherein L is selectedfrom the group consisting of halogen, alkylsulfonyl, substitutedalkylsulfonyl, arylsulfonyl, substituted arylsulfonyl,hetercyclcosulfonyl or trichloroacetimidate
 3. The method of claim 1wherein L is selected from the group consisting of chloro, fluoro,bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl,methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate),p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate),trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl,imidazolesulfonyl and 2,4,6-trichlorophenyl.
 4. The method of claim 1wherein said base is selected from the group consisting of sodiumhydride, methylmagnesium chloride, t-butylmagnesium chloride ormethyl-magnesium fluoride.
 5. The method of claim 1 wherein R_(F) is anacid labile blocking group.
 6. The method of claim 1 wherein Y is ahydrogenolysis sensitive blocking group.
 7. The method of claim 1wherein R_(F) is selected from the group consisting of t-butyl,dimethoxytrityl or tetrahydropyranyl.
 8. The method of claim 1 wherein Yis a nucleotide bound to a solid state support, an oligonucleotide boundto a solid state support, or a solid state support.
 9. The method ofclaim 1 wherein the new first synthon has the structure:


10. The method of claim 1 further comprising repeating said steps (b),(c), and (d) a plurality of times.
 11. The method of claim 1 furthercomprising contacting said f irst synthon with said base in a solventselected from the group consisting of acetonitrile, tetrahydrofuran anddioxane.
 12. The method of claim 1 wherein said reagent used to removeblocking group RF is an acid selected from the group consisting oftrichloroacetic acid, acetic acid or trifluoromethane sulfonic acid. 13.The product of the process of claim
 1. 14. An oligonucleotide comprisinga plurality of nucleoside units linked together via phosphate linkages,wherein: at least one of the nucleoside units is a non-naturallyoccurring nucleoside unit; and at least two of the nucleoside units arelinked via chiral phosphate linkages.
 15. The oligonucleotide of claim14 wherein said chiral phosphate linkages are selected from the groupconsisting of chiral Sp phosphorothioate, chiral Rp phosphorothioiate,chiral Sp alkylphosphonate, chiral Rp alkylphosphonate, chiral Spphosphoamidate, chiral Rp phosphoamidate, chiral Sp chiralphosphotriester or chiral Rp phosphotriester.
 16. The oligonucleotide ofclaim 14 wherein each of the phosphate linkages is a chiral phosphatelinkage.
 17. The oligonucleotide of claim 14 wherein: at least one ofthe nucleoside units has one of the structures:

wherein: Q is O or CHR_(G); R_(A)and R_(B) are H, lower alkyl,substituted lower alkyl, an RNA cleaving moiety, a group which improvesthe pharmacokinetic properties of an oligonucleotide, or a group whichimproves the pharmacodynamic properties of an oligonucleotide; R_(C) isH, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF₃, OCF₃,OCN, O-alkyl, substituted O-alkyl, S-alkyl, substituted S-alkyl, SOMe,SO₂Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂CH═CH₂,OCH═CH₂, OCH₂CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl,polyalkylamino, substituted silyl, an RNA cleaving moiety, a group whichimproves the pharmacodynamic properties of an oligonucleotide, or agroup which improves the pharmacokinetic properties of anoligonucleotide; R_(G) is H, lower alkyl, substituted lower alkyl, anRNA cleaving moiety, a group which improves the pharmacokineticproperties of an oligonucleotide, or a group which improves thepharmacodynamic properties of an oligonucleotide; and B_(X) is anaturally occurring or synthetic nucleoside base.
 18. Theoligonucleotide of claim 17 wherein B_(X) is a pyrimidinyl-1 orpurinyl-9 moiety.
 19. The oligonucleotide of claim 17 wherein B_(X) isadenine, guanine, hypoxanthine, uracil, thymine, cytosine,2-aminoadenine or 5-methylcytosine.
 20. The oligonucleotide of claim 14wherein at least one of the modified nucleotides has one of thestructures:

wherein: G and K are, independently, C or N; J is N or CR₂R₃; R₁ is OHor NH₂; R₂ and R₃ are H, NH, lower alkyl, substituted lower alkyl, loweralkenyl, aralkyl, alkylamino, aralkylamino, substituted alkylamino,heterocycloalkyl, heterocyclo-alkylamino, aminoalkylamino,hetrocycloalkylamino, poly-alkylamino, an RNA cleaving moiety, a groupwhich improves the pharmacokinetic properties of an oligonucleotide, ora group which improves the pharmacodynamic properties of anoligonucleotide; R₄ and R₅ are, independently, H, OH, NH₂, lower alkyl,substituted lower alkyl, substituted amino, an RNA cleaving moiety, agroup which improves the pharmacokinetic properties of anoligonucleotide, or a group which improves the pharmacodynamicproperties of an oligonucleotide; R₆ and R₇ are, independently, H, OH,NH₂, SH, halogen, CONH₂, C(NH)NH₂, C(O)O-alkyl, CSNH₂, CN, C(NH)NHOH,lower alkyl, substituted lower alkyl, substituted amino, an RNA cleavingmoiety, a group which improves the pharmacokinetic properties of anoligonucleotide, or a group which improves the pharmacodynamicproperties of an oligonucleotide; and X is a sugar or a sugar analogmoiety where said sugar analog moiety is a sugar substituted with atleast one substituent comprising an RNA cleaving moiety, a group whichimproves the pharmacodynamic properties of an oligonucleotide, or agroup which improves the pharmacokinetic properties of anoligonucleotide.
 21. The oligonucleotide of claim 20 wherein X has oneof the structures:

wherein: Q is O or CHR_(G); R_(A) and R_(B) are H, lower alkyl,substituted lower alkyl, an RNA cleaving moiety, a group which improvesthe pharmacokinetic properties of an oligonucleotide, or a group whichimproves the pharmacodynamic properties of an oligonucleotide; R_(C) isH, OH, lower alkyl, substituted lower alkyl, F, Cl, Br, CN, CF₃, OCF₃,OCN, O-alkyl, substituted O -alkyl, S-alkyl, substituted S-alkyl, SOMe,SO₂Me, ONO₂, NO₂, N₃, NH₂, NH-alkyl, substituted NH-alkyl, OCH₂CH═CH₂,OCH═CH₂, OCH₂CCH, OCCH, aralkyl, heteroaralkyl, heterocycloalkyl,polyalkylamino, substituted silyl, an RNA cleaving moiety, a group whichimproves the pharmacodynamic properties of an oligonucleotide, or agroup which improves the pharmacokinetic properties of anoligonucleotide; and R_(G) is H, lower alkyl, substituted lower alkyl,an RNA cleaving moiety, a group which improves the pharmacokineticproperties of an oligonucleotide, or a group which improves thepharmacodynamic properties of an oligonucleotide.
 22. Theoligonucleotide of claim 14 wherein the oligonucleotide forms at least aportion of a RNA or DNA sequence.
 23. An oligonucleotide comprising aplurality of nucleoside units linked together by all S_(P)phosphotriester linkages or by all R_(P) phosphotriester linkages. 24.The oligonucleotide of claim 23 wherein said nucleoside units are linkedtogether in a sequence that is antisense to an RNA or DNA sequence. 25.An oligonucleotide comprising a plurality of linked of nucleoside unitslinked together by all S_(P) phosphoramidate linkages or by all R_(P)phosphoamidate linkages.
 26. The oligonucleotide of claim 25 whereinsaid nucleoside units are linked together in a sequence that isantisense to an RNA or DNA sequence.
 27. An oligonucleotide comprisingat least 10 nucleoside units linked together by all S_(P)alkylphosphonate linkages or by all R_(P) alkylphosphonate linkages. 28.The oligonucleotide of claim 27 wherein said alkylphosphonate linkagesare methylphosphonate linkages.
 29. The oligonucleotide of claim 27wherein said nucleoside units are linked together in a sequence that isantisense to an RNA or DNA sequence.
 30. A composition of matter havingthe structure:

wherein: Q is O or CH₂; RA is H, lower alkyl, substituted lower alkyl,an RNA cleaving moiety, a group which improves the pharmacokineticproperties of an oligonucleotide, or a group which improves thepharmacodynamic properties of an oligonucleotide; R_(D) is O, S, methyl,alkoxy, thioalkoxy, amino or substituted amino; R_(E) is O or S; R_(F)is H or a blocking group; R_(X) is H, OH, or a sugar derivatizing group;B_(X) is a nucleoside base, a blocked nucleoside base, a nucleoside baseanalog, or a blocked nucleoside base analog; and L is a leaving group ortogether L and Bx are a 2-3′ or 6-3′ pyrimidine or 8-3′ purinecyclo-nucleoside.
 31. The composition of matter of claim 30 wherein L isselected from the group consisting of halogen, alkylsulfonyl,substituted alkylsulfonyl, arylsulfonyl, substituted arylsulfonyl,hetercyclcosulfonyl or trichloroacetimidate
 32. The composition ofmatter of claim 30 wherein L is selected from the group consisting ofchloro, fluoro, bromo, iodo, p-(2,4-dinitroanilino)benzenesulfonyl,benzene-sulfonyl, methylsulfonyl (mesylate), p-methylbenzenesulfonyl(tosylate), p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate),trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl,imidazolesulfonyl and 2,4,6-trichlorophenyl.
 33. The composition ofmatter of claim 30 wherein R_(X) is H, OH, lower alkyl, substitutedlower alkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, substitutedO-alkyl, S-alkyl, substituted S-alkyl, SOMe, SO₂Me, ONO₂, NO₂, N₃, NH₂,NH-alkyl, substituted NH-alkyl, OCH₂CH═CH₂, OCH═CH₂, OCH₂CCH, OCCH,aralkyl, heteroaralkyl, heterocycloalkyl, polyalkylamino, substitutedsilyl, an RNA cleaving moiety, a group for improving the pharmacodynamicproperties of an oligonucleotide or a group for improving thepharmacokinetic properties of an oligonucleotide.
 34. The composition ofmatter of claim 30 wherein R_(F)is selected from the group consisting ofH, t-butyl, di-methoxytrityl or tetrahydropyranyl.
 35. The compositionof matter of claim 30 wherein B_(X) is a pyrimidinyl-1 or purinyl-9moiety.
 36. The composition of claim 30 wherein B_(X) is adenine,guanine, hypoxanthine, uracil, thymine, cytosine, 2-aminoadenine or5-methylcytosine.
 37. The composition of claim 30 wherein Q is O. 38.The composition of claim 30 having Sp stereochemistry.
 39. Thecomposition of claim 30 having Rp stereochemistry.